“ELISA-CIEF” USING CAPILLARY-BASED MICRODEVICE:
HIGHLY-SENSITIVE ELISA BASED ON
CAPILLARY-ISOELECTRIC FOCUSING OF
ENZYME REACTION PRODUCT
Yuta Uenoyama
1
, Ken Ikegami
2
, Daniel Citterio
2
, Koji Suzuki
2
,
Shun-ichi Funano
1
, Terence G. Henares
1
, Tatsuro Endo
1
, and Hideaki Hisamoto
1
*
1
Osaka Prefecture University, JAPAN,
2
Keio University, JAPAN
ABSTRACT
Sensitivity enhancement of enzyme-linked immunosorbent assay (ELISA) in capillary-based microdevice was
achieved by the concentration of fluorescent final enzyme reaction product using capillary-isoelectric focusing
(CIEF). In order to realize this, fluorescent substrate molecule, which can change its structure into ampholyte ion
after an enzyme reaction, was newly synthesized and used. Approximately seven-fold enhancement of sensitivity
and 1-2 orders of magnitude lower the detection limit were achieved.
KEYWORDS
Capillary isoelectric focusing, Enzyme-linked immunosorbent assay (ELISA), Sensitivity enhancement, Square
capillary
INTRODUCTION
Enzyme-linked immunosorbent assay (ELISA) is an established method of protein analysis using enzyme labeled
antibody. It is widely used for highly sensitive and selective clinical diagnostic tools. However, detection of low
concentration marker proteins still remains as a problem. On the other hand, capillary isoelectric focusing (CIEF) is
a well-known method for the separation and concentration of target molecules possessing isoelectric point, such as
proteins and peptides, by using pH gradient formed inside the capillary. CIEF has an advantage that all target
molecules can be concentrated at a certain point of capillary. Thus, if the final fluorescent product of an enzyme
reaction in ELISA is concentrated, development of highly-sensitive ELISA exceeding the present sensitivity and
detection limit is expected. In this research, design and synthesis of novel fluorescent substrates of alkaline
phosphatase (ALP) which can change its structure into ampholyte ion after an enzyme reaction was carried out and
applied to ELISA-CIEF in capillary-based microdevice.
EXPERIMENT
Synthesis of rhodamine type-ALP substrate (RD-DP) was carried out. ELISA using the synthesized substrates
was performed inside a capture antibody (anti-human IgG)-immobilized square glass capillary (I.D.100m
Capture antibody： Enzyme-antibody： Antigen：
Carrier ampholyte： Substrate： Product：
＋ －
O
NO
2
CO
2
Positively charged site
Negatively-
charged site
Fluorescence
emission site
Rhodamine110
ALP
RD-DP
O
O
O
O
P
HO
O
2
N
O
H
N
O
O
HO
H
N O
O
NO
2
O
P
OH
OH
O
O
O
O
O
P
HO
O
2
N
O
H
N
O
O
HO
H
N O
O
NO
2
O
P
OH
OH
O
O NH H
2
N
OH
O
Figure 1 General concept of ELISA-CIEF on a capillary-based microdevice
PDMS
Square glass capillary
Enzyme
reaction site
Enzyme
reaction site
Figure 2 Newly-synthesized fluorescent substrate, RD-DP, for use in ELISA-CIEF
Reservoirs
16th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
October 28 - November 1, 2012, Okinawa, Japan 978-0-9798064-5-2/μTAS 2012/$20©12CBMS-0001
1735